Interaction of fluorescent delta 5,7,9(11),22-ergostatetraen-3 beta-ol with sterol carrier protein-2.
The fluorescent sterol delta 5,7,9(11)-dehydroergostatetraen-3 beta-ol (dehydroergosterol) was used as an analogue of cholesterol to examine the molecular interaction of purified rat liver sterol carrier protein-2 (SCP-2) with sterol. The binding of dehydroergosterol to SCP-2 was evidenced by light scatter and by fluorescence polarization, lifetime, limiting anisotropy, and rotational relaxation time of dehydroergosterol. In addition, energy transfer efficiency from SCP-2 tryptophan to dehydroergosterol was 96%, indicating that the apparent distance, R, between the SCP-2 tryptophan (energy donor) and the dehydroergosterol (energy acceptor) was 13.7 A. Scatchard binding analysis of light scatter, lifetime, and energy transfer data all indicated a 1:1 molar stoichiometry with Kd = 1.2, 1.6, and 1.3 microM, respectively. SCP-2 enhanced the activity of microsomal acyl-CoA:cholesterol acyltransferase through transfer of [3H]cholesterol from donor palmitoyloleoyl phosphatidylcholine/cholesterol small unilamellar vesicles to rat liver microsomes containing the enzyme. A recently developed fluorescence assay utilizing dehydroergosterol fluorescence polarization (Nemecz, G., Fontaine, R. N., and Schroeder, F. (1988) Biochim. Biophys. Acta 948, 511-521; Nemecz, G., and Schroeder, F. (1988) Biochemistry 27, 7740-7749) was applied to examine the effect of SCP-2 on sterol exchange. In the absence of SCP-2, two spontaneously exchangeable sterol domains were observed in palmitoyloleoyl phosphatidylcholine/sterol (65:35 molar ratio) small unilamellar vesicles. SCP-2 enhanced the rate of exchange of the faster exchanging domain 2-fold. The transfer rate of the more slowly exchangeable sterol domain and the fraction of cholesterol represented by each domain were not affected. These results demonstrate the utility of dehydroergosterol to probe SCP-2 interactions with sterols and are indicative of a physiological role for SCP-2 as a soluble sterol carrier.
Schroeder
F
F
Department of Pharmacology, College of Pharmacy, University of Cincinnati, Ohio 45267-0004.
Butko
P
P
Nemecz
G
G
Scallen
T J
TJ
D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism* / Q000494:pharmacology; D002784:Cholesterol / Q000378:metabolism; D004735:Energy Transfer; D004875:Ergosterol / Q000031:analogs & derivatives* / Q000378:metabolism; D005454:Fluorescence Polarization; D008027:Light; D008081:Liposomes / Q000378:metabolism; D008099:Liver / Q000032:analysis; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology; D010713:Phosphatidylcholines; D010940:Plant Proteins; D011485:Protein Binding; D051381:Rats; D012542:Scattering, Radiation; D002785:Sterol O-Acyltransferase / Q000378:metabolism; D013261:Sterols; D014364:Tryptophan / Q000378:metabolism
D002352:Carrier Proteins; D008081:Liposomes; D010713:Phosphatidylcholines; D010940:Plant Proteins; D013261:Sterols; C072087:sterol carrier proteins; C010905:dehydroergosterol; D014364:Tryptophan; D002784:Cholesterol; D002785:Sterol O-Acyltransferase; C028694:1-palmitoyl-2-oleoylphosphatidylcholine; D004875:Ergosterol